The influence of incubation temperature on offspring traits varies across northern and southern populations of the American alligator (Alligator mississippiensis)

Abstract Maternal provisioning and the developmental environment are fundamental determinants of offspring traits, particularly in oviparous species. However, the extent to which embryonic responses to these factors differ across populations to drive phenotypic variation is not well understood. Here, we examine the contributions of maternal provisioning and incubation temperature to hatchling morphological and metabolic traits across four populations of the American alligator (Alligator mississippiensis), encompassing a large portion of the species' latitudinal range. Our results show that whereas the influence of egg mass is generally consistent across populations, responses to incubation temperature show population‐level variation in several traits, including mass, head length, head width, and residual yolk mass. Additionally, the influence of incubation temperature on developmental rate is greater at northern populations, while the allocation of maternal resources toward fat body mass is greater at southern populations. Overall, our results suggest that responses to incubation temperature, relative to maternal provisioning, are a larger source of interpopulation phenotypic variation and may contribute to the local adaptation of populations.


| INTRODUC TI ON
Developmental plasticity, the expression of alternative phenotypes under different environmental conditions, is a fundamental driver of phenotypic variation across organismal and populationlevel scales.Organismal responses to the developmental environment can be adaptive, neutral, or mal-adaptive (Forsman, 2015;Ghalambor et al., 2007), having important implications for both ecology and evolution (Miner et al., 2005;West-Eberhard, 1989, 2003).For example, when conditions experienced during development provide reliable cues of later life environments, developmental plasticity can be adaptive by maximizing phenotype-environment matching (Nettle & Bateson, 2015;Pfennig, 1990).Alternatively, environments that disrupt normal developmental processes can lead to plastic responses with negative effects on fitness (Barker, 2001;Guillette et al., 1995), while physical constraints on development can give rise to plasticity that is neutral with respect to fitness (Ghalambor et al., 2007;Gotthard & Nylin, 1995).
Regardless of their adaptive value, many embryonic responses to the developmental environment have a heritable, genetic basis and can vary, suggesting they can evolve under novel selective pressures (Pigliucci, 2005).
For example, egg mass is a primary determinant of hatchling mass (Deeming & Birchard, 2007), but incubation temperature has been shown to influence diverse hatchling phenotypes across many species (While et al., 2018).This includes modifying the efficiency by which maternal resources are converted into somatic tissue (Bock et al., 2021;Marshall et al., 2020;Pettersen et al., 2019) and how those resources are allocated to specific phenotypes (Flatt, 2001;Telemeco et al., 2010).However, despite the importance of maternal provisioning and incubation temperature in modifying hatchling phenotypes, the extent to which responses to these factors vary across populations is not well resolved (but see Bodensteiner et al., 2019;Orizaola & Laurila, 2009;Orizaola & Laurila, 2016;Richter-Boix et al., 2015).
When viewed through the lens of Developmental Cost Theory (DCT, Marshall et al., 2020), the influence of incubation temperature on maternal resource use represents a fundamental developmental constraint (Gotthard & Nylin, 1995).According to DCT, the energy required for development can be quantified as the product of development rate and metabolic rate (Pettersen et al., 2019).
Whereas temperature affects both developmental and metabolic rates, differences in their temperature-dependence result in an optimal temperature at which developmental cost is minimized.As a result, environmental temperatures typically encountered by embryos in nature are tightly correlated to species-specific thermal optima that minimize developmental cost (Marshall et al., 2020;Pettersen et al., 2019).Deviations from these optima are predicted to decrease developmental efficiency and result in reduced size, growth, and energy reserves of individuals.Importantly, responses of metabolic rate and developmental rate to temperature can be decoupled (Pettersen, 2020;Williams et al., 2016).Therefore, thermal dependencies of metabolic and/or development rate can evolve independently, allowing selection to modify the temperature at which developmental cost is minimized under novel thermal environments (Pettersen et al., 2023).
In species with broad geographic ranges, divergent climatic conditions have the potential to exert novel selective pressures on traits influenced by the developmental environment (Conover & Schultz, 1995;Kawecki & Ebert, 2004;Merilä et al., 2000;Orizaola & Laurila, 2009).Populations inhabiting high altitudes and latitudes are often exposed to colder temperatures (Angilletta, 2009), which impose novel thermal constraints on development.To compensate, populations can adapt by altering the thermal sensitivity of developmental processes.For instance, in oviparous reptiles, cooler incubation temperatures can result in longer incubation duration.
Embryos from high-altitude and latitude populations compensate by displaying faster development rates when compared to those from lower altitudes or latitudes under identical incubation temperatures (Du, Warner, et al., 2010;Pettersen, 2020), regardless of egg size (Storm & Angilletta Jr, 2007).These opposing effects of genetic and environmental influences on developmental rate, known as counter-gradient variation (Conover & Schultz, 1995), are thought to reduce the cost of development and allow more time for offspring to acquire resources prior to colder, harsher winters (Olsson & Shine, 1997;Pettersen, 2020).Similarly, high-altitude populations of wall lizards (Podacris uralis) have been shown to allocate more maternal resources toward somatic tissue relative to low-altitude populations when raised at a common temperature (Pettersen et al., 2023).
However, our understanding of the extent to which populations vary in how maternal provisioning and incubation temperature shape fitness-related traits in taxonomically diverse species is limited (While et al., 2018).
In the present study, we test whether populations vary in embryonic responses to maternal provisioning and incubation temperature in the American alligator (Alligator mississippiensis).The alligator's latitudinal range extends from southern Florida to northeastern North Carolina (Elsey et al., 2019), providing potential for local adaptation of phenotypic responses to the developmental environment.Few studies have examined variation in nest temperatures across the alligator's range, but comparisons between a northern and southern population did not find significant differences in mean nest temperature (Bock et al., 2020).However, this was based on only 3 years of overlap between populations, and within each year, the mean nest temperature of southern populations was greater than that of northern populations (Bock et al., 2020).Cooler temperatures at northern latitudes would presumably decrease developmental rate, increase the cost of development, and delay hatching dates, reducing time for resource acquisition prior to winter (Olsson & Shine, 1997).Despite these potential differences, there is little information on how responses to the developmental environment vary across the alligator's range.
Like many turtles and some lizards, alligators display temperature-dependent sex determination (TSD), in which thermal signals experienced during a discrete developmental window determine sex, along with additional phenotypic traits (Allsteadt & Lang, 1995;Bock et al., 2021;Kohno et al., 2014;McCoy et al., 2016).Specifically, incubations at warmer, male-promoting temperatures (MPT) reduce developmental costs, producing larger hatchlings with greater residual yolk reserves when compared to incubations at cooler female-promoting temperatures (FPT, Allsteadt & Lang, 1995;Bock et al., 2023, Bock et al., 2021).Recent reports demonstrate that temperature-sensitive traits, including body mass index (BMI) and snout-vent length (SVL), are associated with higher juvenile survival at MPT in the alligator (Bock et al., 2023;Johnson et al., 2023).However, these appear to be context dependent as the relationship between phenotypic traits and survival varies across years (Bock et al., 2023).Nonetheless, given that warmer, MPT appears to be the optimum developmental temperature in this species, we hypothesize that northern populations, presumably exposed to cooler temperatures, will show compensatory responses to incubation temperature.Using a common garden incubation and grow out design, we resolve the relative influences of incubation temperature and maternal provisioning on aspects of developmental cost (hatchling mass and incubation duration), along with other morphological (SVL, tail girth [TG], head length [HL], head width [HW], and BMI) and metabolic (10-day growth, residual yolk mass and fat body mass) traits across populations.We predict that northern populations will display greater mass and developmental and growth rates relative to southern populations at cooler incubation temperatures.
Additionally, we predict that northern populations will have increased residual energy reserves (residual yolk mass and fat body mass), decreasing the need to acquire resources after development prior to winter.1a).After locating nests by helicopter or airboat, all eggs were removed from a nest cavity within 2 weeks of oviposition.Eggs were placed in plastic bus pans with nesting material from natural nests and driven back to the University of Georgia's Savannah River Ecology Laboratory (SREL) in Aiken, SC (within 4-24 h after egg collection), where they were individually weighed and 1-2 eggs from each clutch were staged according to Ferguson (1985) to determine stage at collection.The remaining eggs were transferred into new bus pans with dampened sphagnum moss and kept in commercial incubators (model I36NLC, Percival Scientific, Perry, IA, USA) at 32°C, an intermediate temperature that produces mixed sex ratios (Lang & Andrews, 1994).During this period, eggs were misted twice daily, and bins were rotated once daily within each incubator to limit the effect of intra-incubator temperature variation.Incubator temperatures were also monitored with HOBO TidbiT® v2 Temp Loggers (Onset, Bourne, MA, USA).Embryonic stage 15 (occurring approximately 15 days postoviposition), just prior to the opening of the thermosensitive period of sex determination (McCoy et al., 2015), was predicted based on the stage of eggs at collection and eggs from each population were randomly assigned in a split-clutch design to one of two temperature treatments: a constant MPT (33.5°C) or a constant FPT (29.5°C).Since full clutches were collected for multiple studies, a random subset of 3-10 eggs/clutch/temperature/site were chosen at this time to raise until hatch for this experiment.Throughout the entire incubation period, eggs were continually monitored as above.

| Experimental design and data collection
While it is increasingly noted that constant temperatures may not be reflective of natural nest conditions (Bowden et al., 2014;Hall & Warner, 2020), the temperatures utilized here have been previously examined in the alligator with known effects on hatchling phenotypes, providing a basis with which to compare our results.
Once embryos pierced the eggshell ("pipped"), the date was recorded, and eggs were placed in glass Mason jars (one egg/jar) with damp, sphagnum moss.Embryos were given 48 h to hatch from the egg before being assisted if they did not hatch on their own.Once fully hatched, individuals were weighed using a digital balance (±0.01 g) and SVL and TG were measured using a flexible ruler (±0.1 mm), and HL and HW were measured using calipers (±1 mm).
Hatchlings were then individually marked using unique, numbered toe tags and transported to large, indoor, fiberglass holding tanks where they were held at the SREL aquatic animal facility for 10 days.
The aquatic animal holding facility is a semi-climate controlled building with translucent fiberglass ceilings, mimicking natural light cycles and maintaining temperatures between 21 and 29°C (Johnson et al., 2023;Tuberville et al., 2016).During this period, hatching alligators relied on maternal yolk reserves and were not fed (Allsteadt & Lang, 1995).Water was changed daily (using tap water), and hatchlings were monitored visually twice daily for overall health and sur-

| Statistical analysis
All statistical analyses were conducted in RStudio (R Core Team, 2021, version 4.1.2),and all models were built using the lme4 package (Bates et al., 2015).Model assumptions of residual normality and homoscedasticity were checked visually via residual vs fitted and Q-Q plots, with log transformations made for residual yolk and fat body mass to best meet assumptions.To compare initial egg mass across populations, we used a linear mixed-effects model (LMM) including a fixed-effect of site and random intercepts to control for clutch effects.To determine whether hatch probability or survival to 10-DPH differed across temperatures or sites, we used a generalized linear mixed model with a binomial distribution including temperature, site, and their interaction as fixed effects, including random intercepts of clutches nested within sites.Post-hoc pairwise comparisons were conducted using the emmeans package (Lenth et al., 2023) with Kenward-Roger degrees of freedom and correcting for multiple testing using Tukey's method.
To test for differences in the relative contributions of egg mass and incubation temperature to phenotypic traits across populations, we constructed separate LMMs for every phenotype at each site.In every model, we included fixed effects of egg mass and incubation temperature, while controlling for clutch effects using random intercepts.We then compared model estimates across populations by extracting beta values (i.e., effect size estimates) and 95% confidence intervals (CIs) using the confint function in R. Model beta estimates in which CIs did not overlap zero or another population were considered statistically significant.
To further examine how embryos respond to temperature and maternal provisioning across populations, we used the ggeffects package (Lüdecke, 2018) to predict temperature-specific mean values of each phenotype at a common egg mass, corresponding to the average egg mass across the dataset (x̄ = 82.75g, SD = 9.99), from each population-specific model.By comparing egg mass-corrected mean phenotypes across temperatures and populations, we were able to determine whether populations differed in mean trait values irrespective of egg mass at either or both temperatures and whether variation in the influence of incubation temperature was driven by phenotypic differences at 29.5°C, 33.5°C, or both.Mean values in which 95% CIs did not overlap were considered statistically significant.
Given that populations can also vary in how maternal resources are allocated toward particular phenotypes, we compared ratios of SVL, TG, HL, HW, residual yolk mass, and fat body mass to hatchling mass across populations within and across temperatures using LMMs.For this analysis, we included temperature, site, and their interaction as predictors, along with egg mass as a covariate, controlling for clutches nested within sites using random intercepts.We then compared predicted mean values from the model within and among temperatures across populations using the emmeans package.Values in which CIs did not overlap were considered statistically significant.We used ratios of traits to hatchling mass instead of egg mass for this analysis because there were significant differences in temperature-specific mass across populations (see below), and as a result, differences in the ratio of traits to egg mass would be confounded by population-specific effects of temperature on mass and may not represent differences in the allocation of maternal resources toward specific phenotypes.All figures were created using the R package ggplot2 (Wickham, 2016).

| Morphological traits
Both egg mass and temperature exerted positive effects on hatchling mass across all populations (Table 2).However, whereas the influence of egg mass did not differ across sites (Figure 2a 2).
We next examined the extent to which morphological phenotypes varied across populations within a temperature, including whether differences in the influence of incubation temperature were driven by variation at 33.5°C, 29.5°C, or both by comparing model means under a common egg mass.There were significant differences in trait values between at least two populations for all morphological traits after controlling for egg mass differences, with interpopulation variation in morphological traits occurring primarily at 29.5°C (Appendix).For instance, the influence of incubation temperature on mass of SCE hatchlings was primarily driven by a reduction in mass at 29.5°C relative to the other populations (SCW:  2c).Ratios of morphological traits to hatchling mass showed no significant differences across populations at either temperature.

| Metabolic traits
As with morphological traits, we also examined the effect of egg mass and incubation temperature on metabolic traits across populations.As TA B L E 2 Model results for the influence of incubation temperature and egg mass on phenotypes at each population.Superscripts for each beta value (effect size estimate) denote significant differences (non-overlapping confidence intervals) between sites within each phenotype.Phenotypes in which at least one population comparison was significant are bolded.egg mass increased, ∆mass decreased at the two southern populations, but had no effect in northern populations (Table 2).However, com- There was no significant influence of egg mass on incubation duration at any population, whereas incubation temperature had a negative influence on incubation duration across all sites (Figure 4).The  CI = [44.30,46.08 4).
However, differences across sites within temperatures were not significant (Appendix).

| DISCUSS ION
Patterns of population-level variation in embryonic responses to maternal provisioning and environmental factors have the potential to inform how the developmental environment contributes to evolutionary change.We observed that, generally, the influence of maternal provisioning on hatchling traits did not vary across populations; however, incubation temperature exerted population-specific effects on both morphological and metabolic traits.This may be explained by a constrained relationship between egg mass and hatch mass (Deeming & Birchard, 2007), which is expected to be under strong selection as hatchling mass is often an important component of survival and fitness (Ronget et al., 2018;Stearns, 2000).
Rather than alter this relationship, selection instead tends to act on aspects of maternal allocation, such as egg size and number, to best match population-specific conditions (Angilletta et al., 2004;Sinervo, 1990).On the other hand, responses to incubation temperature may be in part the result of differences in natural nest temperatures across populations (Bock et al., 2020;Du et al., 2019).
Such differences likely select for embryonic responses to temperature that reduce developmental cost and decrease the need to acquire resource prior to colder, harsher winters at northern latitudes (Pettersen et al., 2023).Our results suggest that plastic responses to incubation temperature, but not maternal provisions, vary across populations and have potential to be modified by selection.
The four populations examined in this study encompassed a large proportion of the alligator's latitudinal range, with two populations from the northern extent and two populations from the southern extent.While not statistically significant, we observed a trend for smaller egg masses at the northern populations relative to the southern populations.In crocodylians, egg mass scales with maternal body size (Larriera et al., 2004), and differences in maternal size might underlie population differences observed here.In mammals, animals from high latitudes tend to be larger than those from low latitudes in a pattern known as Bergmann's rule (Blackburn et al., 1999), and while this seems to hold in turtles and birds, it does not in other reptiles, such as squamates (Ashton, 2002;Ashton & Feldman, 2003) and has not been examined in crocodylians.On the other hand, trade-offs between offspring size and number have been shown to vary, with fewer, larger offspring favored in colder environments and later in the reproductive season (Angilletta et al., 2004;Hall et al., 2020).Nonetheless, larger egg sizes at southern populations do not support either of these hypotheses.Alternatively, allometric relationships between maternal size and egg mass can be altered by environmental conditions, such as salinity stress (Murray et al., 2013).Given the lack of information on nesting females here, Apart from latitudinal trends, there were several differences in the influence of incubation temperature between population pairs, specifically between SCE and other populations and primarily driven by temperature's influence on hatchling mass.In alligators, animals incubated at 33.5°C have been previously shown to be larger in mass than those at 29.5°C (Bock et al., 2021), which was upheld across all of our populations.However, at SCE, the reduction of hatchling mass at 29.5°C was particularly pronounced and appeared to drive additional phenotypic differences.Hatchling mass relative to egg mass reflects the efficiency by which maternal resources are converted into hatchling tissue and is likely a product of the energetic cost of embryonic development (Pettersen et al., 2019).The reduction in mass at SCE at 29.5°C relative to the other sites suggests that development at SCE was particularly inefficient at 29.5°C.
Across our populations, SCE is the only coastal site, which may put additional stressors on embryos and breeding females (Albecker & McCoy, 2017).Indeed, the salinity of the incubation environment has been shown previously to have a negative effect on hatchling mass (Bower et al., 2013).However, we only saw an effect at 29.5°C, and while differential responses to incubation temperature under salinity stress have been reported (e.g., Hudak & Dybdahl, 2023), the extent to which egg yolks from SCE have increased salinity, if at all, relative to our other populations is unknown.Interestingly, animals incubated at 29.5°C at SCE also tended to have residual yolk reserves that were larger or equivalent to other populations after controlling for mass.This may be driven by a reduced rate of yolk assimilation during development or may suggest an increased importance of residual yolk mass under cooler temperatures at SCE, despite reduction in overall size (Murphy et al., 2020;Radder et al., 2004).
The lack of latitudinal trends in most of the morphological and metabolic traits examined here suggests that latitude may not be the best or only microclimatic proxy within which to understand variation in responses to the developmental environment, particularly incubation temperature.A similar lack of latitudinal patterns in response to incubation temperature was shown across several populations of painted turtles (Chrysemys picta), another TSD species (Bodensteiner et al., 2019).These results may be driven by microhabitat population differences in temperature that are not represented by latitude.On the other hand, maternal nest site choice can be an important driver of nest temperatures and may vary across populations (Du et al., 2023;Warner & Shine, 2008).This can result in similar nest temperatures despite different environmental temperatures (Bodensteiner et al., 2023)  and the consistency of such effects across years will be particularly informative.
One important component not examined in this study is the role of genetics in shaping trait variation across populations.
Specifically, high gene flow between populations can limit the ability of selection to drive local adaptation, rendering the differences observed across our populations unlikely to have a genetic basis or be adaptive (Kawecki & Ebert, 2004;Stamp & Hadfield, 2020).
Limited information on population structure of alligators exists, but work utilizing microsatellites has shown that populations generally follow an isolation by distance model: genetic differences between FLW and FLE are relatively low, forming a group with other FL and GA populations, but separate from Louisiana and Texas populations (Davis et al., 2002;Ryberg et al., 2002).
Meanwhile, one population examined in SC (Santee Coastal Reserve) was shown to be genetically distinct from both of the latter groups (Davis et al., 2002).These results suggest that there is gene flow between FLW and FLE but limited connectively between them and our northern populations.Given this information, it is likely that FLE and FLW are more closely related genetically than to SCW or SCE, and that genetic distances between FLW and FLE are likely reduced relative to those between SCW and SCE.This aligns with our results as we observed differences in both incubation duration and mass-corrected fat body mass between northern and southern population pairs.Furthermore, while there were no differences between FLE and FLW for any trait, SCE differed from all other populations in response to temperature for mass, showing additional population-specific differences in other traits, usually between SCE and a southern population.However, further work on the genetic structure of these populations is needed to understand the genetic basis of the differences observed, which is critical if they are to be adaptive or modified by selection.

| CON CLUS IONS
Overall, we found variation in developmental plasticity to incubation temperature for morphological and metabolic phenotypes across populations of alligators.In contrast, the influence of maternal provisioning on hatchling traits was mostly consistent across populations.While the adaptive value of variable plastic responses to incubation temperature was not explicitly tested, variation across populations may suggest evolutionary potential.However, the lack of information on environmental differences between populations, differential selective pressures acting on hatchling alligators, and the genetic basis of the differences observed prevent drawing broad

F
Geography and egg size of sampled populations.(a) Map showing the geographic range of the American alligator and sampled populations.(b) Egg mass variation across populations.
vival.At 10-days post-hatch (10-DPH), hatchlings were remeasured, euthanized via cervical severance and pithing, and dissected to obtain residual yolk mass and fat body mass.Phenotypes analyzed included morphological traits of mass, SVL, TG, HL, HW, and body condition (BMI: mass/2*SVL) at hatch, and metabolic traits including incubation duration (measured in days from stage 15 to pip), change in morphological traits between 10-DPH and hatch (∆ mass ∆ BMI, ∆ SVL, ∆ TG), residual yolk mass, and fat body mass.All experiments were approved by the University of Georgia Animal Care and Use Committee (A2021 05-007-Y3-A0), and collections were carried out under permits from the South Carolina Department of Natural Resources (SC-08-2021) and Florida Fish and Wildlife Conservation Commission (SPGS-18-33).
Figure 3b; Appendix).Upon examination of the mass-corrected allocation of maternal resources toward metabolic phenotypes, there were significant differences for both residual yolk mass and fat body mass across populations.Animals from the southern populations tended to allocate more resources toward fat body mass than the northern populations at both 29.5°C (SCW: x̄ = 0.0045, CI = [0.0036,0.0054]; SCE: x̄ = 0.0045, CI = [0.0038,00052]; FLW: x̄ = 0.0063, CI = [0.0055,0.0070]; FLE: x̄ = 0.0061, CI = [0.0054,0.0068]) and 33.5°C (SCW: x̄ = 0.0023, CI = [0.0016,0.0031]; SCE: x̄ = 0.0018, CI = [0.0012,00025]; FLW: x̄ = 0.0030, CI = [0.0024,0.0036]; FLE: x̄ = 0.0033, CI = [0.0026,0.0039]; Figure 3c), and animals from SCE at 29.5°C allocated more resources toward residual yolk mass relative to SCW and FLE (SCW: x̄ = 0.0129, CI = [0.0078,0.0179]; SCE: influence of temperature was greater at the northern populations than at the southern populations (SCW: β = −14.16,CI = [−15.22,−13.15];SCE: β = −13.07,CI = [−13.51,−12.62];FLW: β = −10.86,CI = [−11.95,−9.71]; FLE: β = −11.08,CI = [−12.02,−10.14]), driven by comparatively shorter incubation periods at 33.5°C (SCW: x̄ = 45.19, it remains unknown whether differences in egg size are the result of variation in maternal size across populations (maximum size or age at reproduction), population-specific allometric relationships, or differences in maternal allocation and is an interesting area of future research.We hypothesized that northern populations would show evidence of adaptation to cooler environments by altering embryonic responses to temperature, resulting in faster development and increased mass and growth at cool incubation temperatures.However, only a few traits showed evidence of latitudinal patterns.We found that incubation duration was more strongly influenced by incubation temperature at the northern populations relative to the southern populations.Specifically, embryos from northern populations developed slightly slower at cooler temperatures and faster at warmer temperatures compared to southern populations.Latitudinal differences in incubation duration have been shown in several species and generally follow one of two patterns: co-gradient variation, in which cooler population development more slowly relative to warmer populations and counter-gradient variation, in which cooler populations development more quickly than warmer populations (Conover & Schultz, 1995; Pettersen, 2020).While our differences within temperatures were not significant, they followed patterns of both co-gradient variation (at 29.5°C) and counter-gradient variation (at 33.5°C), which only partially support our predictions.Similar results F I G U R E 4 Population variation in the influence of incubation temperature on incubation duration.Beta values are effect size estimates, and error bars represent 95% confidence intervals.have been shown in Asian pond turtles (Mauremys mutica; Zhao et al., 2015) and may suggest that the mechanisms responsible for variation in incubation duration across populations are temperature specific.Alternatively, increased plasticity of developmental rate at northern populations may allow embryos to take advantage of warm conditions when they do arise under natural thermal regimes, reducing development time and the cost of development and resulting in earlier hatching.Additional experiments incorporating more incubation treatments and populations are needed to more completely discern how the relationship between temperature and developmental rate differs across populations as well as the underlying mechanisms responsible.We also observed that southern populations tended to allocate more resources toward fat body mass than northern populations at both incubation temperatures, opposite our predictions.The role of the fat body in alligators is not known, and further work examining its function, including how fat body size/mass early in life might impact survival and later life fitness, is needed to more fully appreciate the potential consequences of this pattern.
conclusions.Determining the causes of these differences, including the developmental mechanisms involved, would provide important insight into how components of the developmental environment and embryonic responses to them influence intraspecific variation and may contribute to adaptive evolutionary change.AUTH O R CO NTR I B UTI O N S Christopher R. Smaga: Conceptualization (equal); formal analysis (lead); investigation (lead); writing -original draft (lead).Samantha L. Bock: Investigation (equal); writing -review and editing (equal).Josiah M. Johnson: Investigation (equal); writing -review and editing (equal).Thomas Rainwater: Investigation (supporting); writing -review and editing (equal).Randeep Singh: Investigation (supporting); writing -review and editing (equal).Vincent Deem: Investigation (supporting); writing -review and editing (equal).Andrew Letter: Investigation (supporting); writing -review and editing (equal).Arnold Brunell: Investigation (supporting); writing -review and editing (equal).Benjamin Parrott: Conceptualization (equal); funding acquisition (lead); investigation (equal); resources (lead); supervision (lead); writing -review and editing (equal).ACK N OWLED G M ENTS This work was made possible through support from the National Science Foundation (Award 1754903 (B.B.P)) and the United States Department of Energy Cooperative Agreement (DE-EM0004391).We are also grateful to the South Carolina Department of Natural Resources and Tom Yawkey Wildlife Center staff for permitting and assistance in the field.We thank members of the Parrott Lab for insightful conversations regarding this manuscript.This paper represents technical contribution number 7209 of the Clemson University Experimental Station.This article has earned an Open Data badge for making publicly available the digitally-shareable data necessary to reproduce the reported results.The data is available at https:// doi.org/ 10. 5061/ dryad.280gb 5mvk.DATA AVA I L A B I L I T Y S TAT E M E N TData for the manuscript are available on dryad (https:// datad ryad.org/ stash/ share/ Ee6Q w q0ccV 3fJku 7nMFd kJySn mvDiD oMGuy ug-J75Lo ).D I SCL A I M ERThis report was prepared as an account of work sponsored by an agency of the US Government.Neither the US Government nor any agency thereof, nor any of their employees, make any warranty, express or implied, or assume any legal liability or responsibility for the accuracy, completeness, or usefulness of any information, apparatus, product, or process disclosed, or represent that its use would not infringe privately owned rights.Reference herein to any specific commercial product, process, or service by trade name, trademark, manufacturer, or otherwise does not necessarily constitute or imply its endorsement recommendation, or favoring by the US Government or any agency thereof.The views and opinions of authors expressed herein do not necessarily state or reflect those of the US Government or any agency thereof.O RCI DChristopher R. Smaga https://orcid.org/0000-0002-1372-5276RE FE R E N C E S